Bottom Line:
This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown.Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes.Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

ABSTRACTEpigenetic changes are thought to lead to alterations in the property of cells, such as differentiation potential. Neural precursor cells (NPCs) differentiate only into neurons in the midgestational brain, yet they become able to generate astrocytes in the late stage of development. This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown. Here we analyzed DNA methylation changes in mouse NPCs between the mid-(E11.5) and late (E14.5) stage of development by a genome-wide DNA methylation profiling method using microarrays and found that many astrocytic genes are demethylated in late-stage NPCs, enabling the cell to become competent to express these genes. Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes. Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

pone-0003189-g005: Expression of the genes hypomethylated both in E14.5 NPCs and astrocytes compared to E11.5 NPCs.Among hypomethylated genes, genes which showed upregulation in E14.5 NPCs or astrocytes compared to E11.5 NPCs are shown. Expression is calculated from data obtained by expression microarray analysis and presented as fold change compared to E11.5 NPCs. Gfap is indicated by an arrow.

Mentions:
Expression changes during the differentiation potential switch of NPCs were analyzed by comparing mid-(E11.5) and late (E14.5)-stage NPCs, and astrocytes by expression microarray analysis (Fig. 4, Table S2). We found that 49% of the probes upregulated in E14.5 NPCs compared to E11.5 NPCs were also upregulated in astroctyes (Fig. 3B). This is smaller than the ratio of hypomethylation where 80% of the probes hypomethylated in E14.5 NPCs were also hypothylated in astrocytes (Fig. 2C). This implies that astrocyte-specific genes, including Gfap, Aldoc, and Kcnj10 (Kir4.1), are demethylated at E14.5 NPCs, while they are not expressed until cells differentiate into astrocytes. To examine this hypothesis, we analyzed the expression of genes hypomethylated both in E14.5 NPCs and astrocytes. Among these genes, those that showed upregulation in E14.5 NPCs or astrocytes compared to E11.5 NPCs are shown in Fig. 5. Of these, 64% of the genes, including astrocyte-specific marker Gfap, showed more than twice the expression level in astrocytes compared to E14.5 NPCs. Thus, genes already demethylated, such as Gfap, Aldoc, and Kcnj10 (Kir4.1), were competent but not highly expressed in E14.5 NPCs, suggesting that their expression appears after differentiation into astrocytes (Fig. 6). Astrocyte-specific genes probably require the expression and/or activation of astrocyte-inducing transcription factors, such as STAT3, to be effectively expressed (Fig. 6).

pone-0003189-g005: Expression of the genes hypomethylated both in E14.5 NPCs and astrocytes compared to E11.5 NPCs.Among hypomethylated genes, genes which showed upregulation in E14.5 NPCs or astrocytes compared to E11.5 NPCs are shown. Expression is calculated from data obtained by expression microarray analysis and presented as fold change compared to E11.5 NPCs. Gfap is indicated by an arrow.

Mentions:
Expression changes during the differentiation potential switch of NPCs were analyzed by comparing mid-(E11.5) and late (E14.5)-stage NPCs, and astrocytes by expression microarray analysis (Fig. 4, Table S2). We found that 49% of the probes upregulated in E14.5 NPCs compared to E11.5 NPCs were also upregulated in astroctyes (Fig. 3B). This is smaller than the ratio of hypomethylation where 80% of the probes hypomethylated in E14.5 NPCs were also hypothylated in astrocytes (Fig. 2C). This implies that astrocyte-specific genes, including Gfap, Aldoc, and Kcnj10 (Kir4.1), are demethylated at E14.5 NPCs, while they are not expressed until cells differentiate into astrocytes. To examine this hypothesis, we analyzed the expression of genes hypomethylated both in E14.5 NPCs and astrocytes. Among these genes, those that showed upregulation in E14.5 NPCs or astrocytes compared to E11.5 NPCs are shown in Fig. 5. Of these, 64% of the genes, including astrocyte-specific marker Gfap, showed more than twice the expression level in astrocytes compared to E14.5 NPCs. Thus, genes already demethylated, such as Gfap, Aldoc, and Kcnj10 (Kir4.1), were competent but not highly expressed in E14.5 NPCs, suggesting that their expression appears after differentiation into astrocytes (Fig. 6). Astrocyte-specific genes probably require the expression and/or activation of astrocyte-inducing transcription factors, such as STAT3, to be effectively expressed (Fig. 6).

Bottom Line:
This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown.Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes.Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.

ABSTRACTEpigenetic changes are thought to lead to alterations in the property of cells, such as differentiation potential. Neural precursor cells (NPCs) differentiate only into neurons in the midgestational brain, yet they become able to generate astrocytes in the late stage of development. This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown. Here we analyzed DNA methylation changes in mouse NPCs between the mid-(E11.5) and late (E14.5) stage of development by a genome-wide DNA methylation profiling method using microarrays and found that many astrocytic genes are demethylated in late-stage NPCs, enabling the cell to become competent to express these genes. Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes. Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.